scholarly journals A Retinoblastoma Orthologue Is Required for the Sensing of a Chalone in Dictyostelium discoideum

2014 ◽  
Vol 13 (3) ◽  
pp. 376-382 ◽  
Author(s):  
Deenadayalan Bakthavatsalam ◽  
Michael J. V. White ◽  
Sarah E. Herlihy ◽  
Jonathan E. Phillips ◽  
Richard H. Gomer
Keyword(s):  
Cell Proliferation ◽  
Dictyostelium Discoideum ◽  
Accumulation Rate ◽  
Protein Accumulation ◽  
Wild Type ◽  
Spore Viability ◽  
Content Type ◽  
Multicellular Development ◽  
Normal Mass ◽  
Inhibit Cell Proliferation

ABSTRACT Retinoblastoma-like proteins regulate cell differentiation and inhibit cell proliferation. The Dictyostelium discoideum retinoblastoma orthologue RblA affects the differentiation of cells during multicellular development, but it is unclear whether RblA has a significant effect on Dictyostelium cell proliferation, which is inhibited by the secreted proteins AprA and CfaD. We found that rblA − cells in shaking culture proliferate to a higher density, die faster after reaching stationary density, and, after starvation, have a lower spore viability than wild-type cells, possibly because in shaking culture, rblA − cells have both increased cytokinesis and lower extracellular accumulation of CfaD. However, rblA − cells have abnormally slow proliferation on bacterial lawns. Recombinant AprA inhibits the proliferation of wild-type cells but not that of rblA − cells, whereas CfaD inhibits the proliferation of both wild-type cells and rblA − cells. Similar to aprA − cells, rblA − cells have a normal mass and protein accumulation rate on a per-nucleus basis, indicating that RblA affects cell proliferation but not cell growth. AprA also functions as a chemorepellent, and RblA is required for proper AprA chemorepellent activity despite the fact that RblA does not affect cell speed. Together, our data indicate that an autocrine proliferation-inhibiting factor acts through RblA to regulate cell density in Dictyostelium , suggesting that such factors may signal through retinoblastoma-like proteins to control the sizes of structures such as developing organs or tumors.

scholarly journals The ROCO Kinase QkgA Is Necessary for Proliferation Inhibition by Autocrine Signals in Dictyostelium discoideum

2010 ◽  
Vol 9 (10) ◽  
pp. 1557-1565 ◽  
Author(s):  
Jonathan E. Phillips ◽  
Richard H. Gomer
Keyword(s):  
Cell Proliferation ◽  
Dictyostelium Discoideum ◽  
Fluorescent Protein ◽  
Signal Transduction Pathway ◽  
Protein Accumulation ◽  
Proliferation Inhibition ◽  
Wild Type ◽  
Content Type ◽  
Multicellular Development ◽  
Green Fluorescent

ABSTRACT AprA and CfaD are secreted proteins that function as autocrine signals to inhibit cell proliferation in Dictyostelium discoideum. Cells lacking AprA or CfaD proliferate rapidly, and adding AprA or CfaD to cells slows proliferation. Cells lacking the ROCO kinase QkgA proliferate rapidly, with a doubling time 83% of that of the wild type, and overexpression of a QkgA-green fluorescent protein (GFP) fusion protein slows cell proliferation. We found that qkgA − cells accumulate normal levels of extracellular AprA and CfaD. Exogenous AprA or CfaD does not slow the proliferation of cells lacking qkgA, and expression of QkgA-GFP in qkgA − cells rescues this insensitivity. Like cells lacking AprA or CfaD, cells lacking QkgA tend to be multinucleate, accumulate nuclei rapidly, and show a mass and protein accumulation per nucleus like those of the wild type, suggesting that QkgA negatively regulates proliferation but not growth. Despite their rapid proliferation, cells lacking AprA, CfaD, or QkgA expand as a colony on bacteria less rapidly than the wild type. Unlike AprA and CfaD, QkgA does not affect spore viability following multicellular development. Together, these results indicate that QkgA is necessary for proliferation inhibition by AprA and CfaD, that QkgA mediates some but not all of the effects of AprA and CfaD, and that QkgA may function downstream of these proteins in a signal transduction pathway regulating proliferation.

scholarly journals PakD, a Putative p21-Activated Protein Kinase in Dictyostelium discoideum, Regulates Actin

2013 ◽  
Vol 13 (1) ◽  
pp. 119-126 ◽  
Author(s):  
Miguel Garcia ◽  
Sibnath Ray ◽  
Isaiah Brown ◽  
Jon Irom ◽  
Derrick Brazill
Keyword(s):  
Protein Kinase ◽  
Actin Cytoskeleton ◽  
Dictyostelium Discoideum ◽  
Cell Function ◽  
Actin Dynamics ◽  
Wild Type ◽  
Content Type ◽  
Multicellular Development ◽  
Actin Organization ◽  
Cellular Processes

ABSTRACT Proper regulation of the actin cytoskeleton is essential for cell function and ultimately for survival. Tight control of actin dynamics is required for many cellular processes, including differentiation, proliferation, adhesion, chemotaxis, endocytosis, exocytosis, and multicellular development. Here we describe a putative p21-activated protein kinase, PakD, that regulates the actin cytoskeleton in Dictyostelium discoideum . We found that cells lacking pakD are unable to aggregate and thus unable to develop. Compared to the wild type, cells lacking PakD have decreased membrane extensions, suggesting defective regulation of the actin cytoskeleton. pakD − cells show poor chemotaxis toward cyclic AMP (cAMP) but normal chemotaxis toward folate, suggesting that PakD mediates some but not all chemotaxis responses. pakD − cells have decreased polarity when placed in a cAMP gradient, indicating that the chemotactic defects of the pakD − cells may be due to an impaired cytoskeletal response to cAMP. In addition, while wild-type cells polymerize actin in response to global stimulation by cAMP, pakD − cells exhibit F-actin depolymerization under the same conditions. Taken together, the results suggest that PakD is part of a pathway coordinating F-actin organization during development.

scholarly journals CbfA, the C-Module DNA-Binding Factor, Plays an Essential Role in the Initiation of Dictyostelium discoideum Development

2004 ◽  
Vol 3 (5) ◽  
pp. 1349-1358 ◽  
Author(s):  
Thomas Winckler ◽  
Negin Iranfar ◽  
Peter Beck ◽  
Ingo Jennes ◽  
Oliver Siol ◽  
...  
Keyword(s):  
Dna Binding ◽  
Cell Density ◽  
Dictyostelium Discoideum ◽  
Ectopic Expression ◽  
Development Program ◽  
Cell Physiology ◽  
Dependent Manner ◽  
Wild Type ◽  
Gene Encoding ◽  
Multicellular Development

ABSTRACT We recently isolated from Dictyostelium discoideum cells a DNA-binding protein, CbfA, that interacts in vitro with a regulatory element in retrotransposon TRE5-A. We have generated a mutant strain that expresses CbfA at <5% of the wild-type level to characterize the consequences for D. discoideum cell physiology. We found that the multicellular development program leading to fruiting body formation is highly compromised in the mutant. The cells cannot aggregate and stay as a monolayer almost indefinitely. The cells respond properly to prestarvation conditions by expressing discoidin in a cell density-dependent manner. A genomewide microarray-assisted expression analysis combined with Northern blot analyses revealed a failure of CbfA-depleted cells to induce the gene encoding aggregation-specific adenylyl cyclase ACA and other genes required for cyclic AMP (cAMP) signal relay, which is necessary for aggregation and subsequent multicellular development. However, the cbfA mutant aggregated efficiently when mixed with as few as 5% wild-type cells. Moreover, pulsing cbfA mutant cells developing in suspension with nanomolar levels of cAMP resulted in induction of acaA and other early developmental genes. Although the response was less efficient and slower than in wild-type cells, it showed that cells depleted of CbfA are able to initiate development if given exogenous cAMP signals. Ectopic expression of the gene encoding the catalytic subunit of protein kinase A restored multicellular development of the mutant. We conclude that sensing of cell density and starvation are independent of CbfA, whereas CbfA is essential for the pattern of gene expression which establishes the genetic network leading to aggregation and multicellular development of D. discoideum.

scholarly journals Arrestins function in cAR1 GPCR-mediated signaling and cAR1 internalization in the development of Dictyostelium discoideum

2014 ◽  
Vol 25 (20) ◽  
pp. 3210-3221 ◽  
Author(s):  
Xiumei Cao ◽  
Jianshe Yan ◽  
Shi Shu ◽  
Joseph A. Brzostowski ◽  
Tian Jin
Keyword(s):  
Dictyostelium Discoideum ◽  
Protein C ◽  
Wild Type ◽  
Multicellular Development ◽  
Membrane Recruitment ◽  
Camp Receptor ◽  
Erk2 Activation ◽  
G Protein Coupled ◽  
Camp Stimulation ◽  
Signaling Circuit

Oscillation of chemical signals is a common biological phenomenon, but its regulation is poorly understood. At the aggregation stage of Dictyostelium discoideum development, the chemoattractant cAMP is synthesized and released at 6-min intervals, directing cell migration. Although the G protein–coupled cAMP receptor cAR1 and ERK2 are both implicated in regulating the oscillation, the signaling circuit remains unknown. Here we report that D. discoideum arrestins regulate the frequency of cAMP oscillation and may link cAR1 signaling to oscillatory ERK2 activity. Cells lacking arrestins (adcB−C−) display cAMP oscillations during the aggregation stage that are twice as frequent as for wild- type cells. The adcB−C− cells also have a shorter period of transient ERK2 activity and precociously reactivate ERK2 in response to cAMP stimulation. We show that arrestin domain–containing protein C (AdcC) associates with ERK2 and that activation of cAR1 promotes the transient membrane recruitment of AdcC and interaction with cAR1, indicating that arrestins function in cAR1-controlled periodic ERK2 activation and oscillatory cAMP signaling in the aggregation stage of D. discoideum development. In addition, ligand-induced cAR1 internalization is compromised in adcB−C− cells, suggesting that arrestins are involved in elimination of high-affinity cAR1 receptors from cell surface after the aggregation stage of multicellular development.

scholarly journals Paxillin and Phospholipase D Interact To Regulate Actin-Based Processes in Dictyostelium discoideum

2011 ◽  
Vol 10 (7) ◽  
pp. 977-984 ◽  
Author(s):  
Jelena Pribic ◽  
Rebecca Garcia ◽  
May Kong ◽  
Derrick Brazill
Keyword(s):  
Dictyostelium Discoideum ◽  
Phospholipase D ◽  
Actin Polymerization ◽  
Genetic Interaction ◽  
Actin Reorganization ◽  
Content Type ◽  
Multicellular Development ◽  
Substrate Adhesion ◽  
Cell Substrate ◽  
Cell Substrate Adhesion

ABSTRACT The actin cytoskeleton forms a membrane-associated network whose proper regulation is essential for numerous processes, including cell differentiation, proliferation, adhesion, chemotaxis, endocytosis, exocytosis, and multicellular development. In this report, we show that in Dictyostelium discoideum , paxillin (PaxB) and phospholipase D (PldB) colocalize and coimmunoprecipitate, suggesting that they interact physically. Additionally, the phenotypes observed during development, cell sorting, and several actin-required processes, including cyclic AMP (cAMP) chemotaxis, cell-substrate adhesion, actin polymerization, phagocytosis, and exocytosis, reveal a genetic interaction between paxB and pldB , suggesting a functional interaction between their gene products. Taken together, our data point to PldB being a required binding partner of PaxB during processes involving actin reorganization.

scholarly journals Spatial and temporal expression of the Dictyostelium discoideum G alpha protein subunit G alpha 2: expression of a dominant negative protein inhibits proper prestalk to stalk differentiation.

1994 ◽  
Vol 5 (1) ◽  
pp. 7-16 ◽  
Author(s):  
F Carrel ◽  
S Dharmawardhane ◽  
A M Clark ◽  
J A Powell-Coffman ◽  
R A Firtel
Keyword(s):  
Dictyostelium Discoideum ◽  
Receptor Activation ◽  
Dominant Negative ◽  
Wild Type ◽  
Protein Subunit ◽  
Multicellular Development ◽  
Stage Of Development ◽  
Downstream Effectors ◽  
Camp Receptor

Previous results have shown that the G alpha protein subunit G alpha 2 is required for aggregation in Dictyostelium discoideum and is essential for coupling cell-surface cAMP receptors to downstream effectors in vivo during this stage of development. G alpha 2 expresses at least four distinct transcripts that are differentially regulated during development; two of the transcripts are expressed exclusively in the multicellular stages and their expression is restricted to prestalk cells. We partially dissected the G alpha 2 promoter and identified a component that is expressed exclusively during the multicellular stages using luciferase gene fusions. When this promoter region is coupled to lacZ, beta-gal expression is restricted to the multicellular stages and localized in prestalk cells with a pattern similar to that of the ecmA prestalk-specific promoter. We show that expression in wild-type cells of the G alpha 2 mutant protein [G alpha 2(G206T)] during the early stages of development blocks aggregation and cAMP-mediated activation of adenylyl cyclase and guanylyl cyclase, suggesting it functions as a dominant negatively active G alpha subunit. When this mutant G alpha protein is expressed from the ecmA prestalk-specific promoter, abnormal stalk differentiation during culmination is observed. Expression of the mutant G alpha 2 from the SP60 prespore promoter or wild-type G alpha 2 from either the ecmA or the SP60 promoter results in no detectable phenotype. The results suggest that G alpha 2 plays an essential role during the culmination stage in prestalk cells and may mediate cAMP receptor activation of these processes during multicellular development.

scholarly journals Unconventional Secretion of AcbA in Dictyostelium discoideum through a Vesicular Intermediate

2010 ◽  
Vol 9 (7) ◽  
pp. 1009-1017 ◽  
Author(s):  
Matthew Cabral ◽  
Christophe Anjard ◽  
Vivek Malhotra ◽  
William F. Loomis ◽  
Adam Kuspa
Keyword(s):  
Cell Surface ◽  
Dictyostelium Discoideum ◽  
Wild Type ◽  
Content Type ◽  
Sensitive Factor ◽  
Late Step ◽  
Unconventional Secretion ◽  
Membrane Bound ◽  
Acyl Coenzyme A ◽  
Mutant Cells

ABSTRACT The acyl coenzyme A (CoA) binding protein AcbA is secreted unconventionally and processed into spore differentiation factor 2 (SDF-2), a peptide that coordinates sporulation in Dictyostelium discoideum. We report that AcbA is localized in vesicles that accumulate in the cortex of prespore cells just prior to sporulation. These vesicles are not observed after cells are stimulated to release AcbA but remain visible after stimulation in cells lacking the Golgi reassembly stacking protein (GRASP). Acyl-CoA binding is required for the inclusion of AcbA in these vesicles, and the secretion of AcbA requires N-ethylmaleimide-sensitive factor (NSF). About 1% of the total cellular AcbA can be purified within membrane-bound vesicles. The yield of vesicles decreases dramatically when purified from wild-type cells that were stimulated to release AcbA, whereas the yield from GRASP mutant cells was only modestly altered by stimulation. We suggest that these AcbA-containing vesicles are secretion intermediates and that GRASP functions at a late step leading to the docking/fusion of these vesicles at the cell surface.

Adhesion barriers inhibit cell proliferation, adhesion and invasion of primary endometriosis cells

2014 ◽  
Vol 74 (S 01) ◽  
Author(s):  
SP Renner ◽  
MW Beckmann ◽  
S Burghaus ◽  
R Strick ◽  
PL Strissel ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Adhesion And Invasion ◽  
Inhibit Cell Proliferation

scholarly journals Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

2020 ◽  
Vol 65 (1) ◽  
pp. e01948-20
Author(s):  
Dalin Rifat ◽  
Si-Yang Li ◽  
Thomas Ioerger ◽  
Keshav Shah ◽  
Jean-Philippe Lanoix ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Clinical Evidence ◽  
Clinical Samples ◽  
Loss Of Function ◽  
Wild Type ◽  
Content Type ◽  
Solid Media ◽  
High Level ◽  
Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

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